IgM pentamer (900 kDa) is a potential disadvantage in that dist1'ibution of the large
`molecule may be restricted. However, it is possible to produce Fab fragments oflgM.
`Coupling in vitro immunization with the standard fusion procedure io this
`study led to a fusion frequency of I 00% with all hybrid lines secreting antibodies.
`Despite the success of the fusion, none of the hybridomas secreted anti-receptor
`immunoglobulins, as assessed by the receptor binding assay used.
`In summary, no sera positive for receptor binding activity was found following
`in vivo immunization with crude membrane preparations. in vitro immunization was
`undertaken because the technique offered a number of potential advantages, but a
`successful fusion did not lead to hybridomas secreting supematants with receptor
`binding activity.
`
`References
`
`BOSS, B. D. (1984). An impl'oved in vitro immunization procedure for the production of monoclonal
`antibodies against neural and other antigens. Brain Res., 291, I 93-t 96.
`
`BOSS, B. D. (1986). An improved in vitro imn1uni:r..ation procedure for the production of monoclonal
`antibodies. Methods Enzymol., 121, 27-33.
`
`CHANTRY, A., LEIGHTON, a. & DAY, A. J. (1991). Cross-reactivity of amylio with calcitonin(cid:173)
`gene-related peptide binding sites in rat liver and skoletal muscle membranes. Biochem.J., 277, 139-
`143.
`
`DEBLASI, A., O'REILLY, K. & MOTULSKY, M. J, ( 1989). Calculating receptor number from
`binding experiments using same compound as radioligand and competitor. Trends Pharmacol. Sci .•
`10, 227-229.
`
`GALFRE, G. & MILSTEIN, C. (1981). Preparation of monoclonal antibodies: strategies and
`procedures. Methods Enzymol., 73, 3-46.
`
`GODING, J. W. ( 1986). Monoclonal anlibodies: Prfnclples and Practice. London: Academic Press.
`
`HAWKES, R. (1986). The dot immunobinding assay. Methods E11zymol., 121, 484-491.
`
`HENKE, H., SIGRIST, S., LANG, W., SCHNEIDER, J. & FISCHER, J. A. (1987). Comparison of
`binding sites for the calcitonin gene-related peptides l and II in man. Brain Res., 410, 404-408.
`
`INAGAKI, S., KITO, S., KUBOTA, Y., GfRGIS, S., HILLYARD, C. J. & MACINTYRE. I. (1986).
`
`130
`
`

`

`Autoradiographic localization of calcitonin gene-related peptide binding sites in hwnan and rat
`brains. Brain Res., 374, 287-298.
`
`NAKAMUTA, H., FUKUDA, Y., KOIDA, M., FUID, N., OTAKA, A., FVNAKOSHI, S., YAJIMA,
`II., MITSUYASU, N. & ORLOWSKI, R. C. (I 986). Binding sites of calcltonin gene-related
`peptide (CGRP): abundant occurrence in visceral organs. Jpn.lPharmaco/., 42, 17 5-180.
`
`READING, C. L. (I 982). Theory and methods for immunization in culture and monoclonal antibody
`production. J.Jmmuno/.Methods, 53, 261-291.
`
`READING, C. L. (1986). In vitro inmunization for the production of antigen specific lymphocyte
`hybridomas. Methods Enzymol., 121, 18-27.
`
`ROTH, R. A. & MORGAN, D. 0. {1985). Monoclonal antibodies to tJ,e insulin receptor.
`Pharmacol. Ther., 28, 1- 16.
`
`SOOS, M. A., SIDDLE, K., BARON, M. D., HEWARD, J. M., LUZ[O, J. P., BELLATJN, J. &
`LENNOX, E. S. (1986). Monoclonal antibodies reacting with multiple epitopes on the human
`insulin receptor. 8iochem.J., 235, 199-208.
`
`STANGL, 0., MUFF, R., SCHMOLCK, C. & FISCHER, J. A. (1993). Photoaffinity labeling of rat
`calcitonin gene-related peptide receptors and adenylate cyclase activation: identification of receptor
`subtypes. Endocrinology, 132, 744-750,
`
`WfMALA WANSA, S. J. & EL-KHOLY, A. A. (1993). Comparative study of distribution and
`biochemical characterization of brain calcitonin gene-related peptide receptors in five different
`species. Neuroscience, 54, 513-5 19.
`
`131
`
`

`

`CHAPTERS
`
`Characterization of CGRP receptor binding of monoclonal
`antibodies raised by an auto-anti-idiotypic approach or by
`immunization with purified CGRP receptor
`
`6.1. Introduction
`
`The anti-idiotypic approach to the production of anti-receptor antibodies is based on
`the idiotypic-anti-idiotypic network hypothesis (Jcme, 1974; Jcme et al., 1982).
`Idiotopes are antigenic determinants
`located
`in
`the variable
`regions of
`immunoglobuJin molecules. The network hypothesis was formulated in recognition of
`the dual character of immu.noglobulin molecules which consist of two distinct entities:
`combining site, or paratope, which interacts with antigens (epitopes), and idiotopes,
`which can be recognized by the paratopes carried on other antibody molecules. The
`anti-idiotypic response is heterogeneous and the network hypothesis attempts to
`classify the different kinds of idiotype-anti-icliotype interactions. The major criterion
`used to distinguish the different anti-idiotypic antibodies is the location of the target
`icliotope to which the anti-icliotype binds in relation to the antigen combining site
`(paratope). The location of the target idiotope can be mapped using the relevant
`antigen or hapten as the inhibitor in the binding of anti-idiotope to idiotope. If antigen
`no inhibition is observed, the target idiotope is assumed to be distant from the
`paratope, and perhaps located in a framework region (Bona & Kohler, 1984). Such
`antigen non-inhibitable anti-idiotypes are called Ab2a. If antigen inhibits the
`idiotope-anti-idiotope interaction, the target idiotope is believed to be in or near the
`paratope. Such anti-idiotypic antibodies, whose idiotopes cross-react with foreign
`epitopes, are postulated to bear the internal image of the original antigen and are
`classified as Ab2l3. Other anti-idiotypes have been classified, for exam.pie, Ab2y
`which are not internal image-bearing but nevertheless antigcn-inhibitable through
`steric interference (Bona & Kohler, 1984).
`The above classification of anti-idiotypes is probably simplistic and concepts
`are being revised (Kohler et al., 1989; Erlanger, 1989). However, the concept of
`internal image bearing anti-idiotypic antibodies has offered the possibility of
`developing anti-receptor antibodies by immunizing with anti-ligand antibodies and,
`therefore, bypass the need to purify the receptor. The network hypothesis further
`predicts that immunization with a ligand would lead to anti-ligand antibodies (Ab1)
`which would, in turn, lead to anti-idiotypic antibodies (Ab2) in the same animal. Jt
`should be possible to screen for internal image-bearing Ab2P anti-idiotypes which
`mimic the ligand and bind to receptor.
`
`132
`
`

`

`The anti-idjotypic route to anti-receptor antibodies was first reported by Sege
`& Peterson (1978) who demonstrated that anti-idiotypic antibodies raised against
`antibodies to insulin could reproduce certain biological actions of the hormone itself
`upon binding to the insulin receptors of rat thymocytes. The findings were confirmed
`by Schechter et al. (1982) who also showed for the first time that immunization of
`mice with insulin led to the development of not only anti-insulin antibodies but also
`autologous anti-idiotypic anti-insulin receptor antibodies. These findings suggested
`that a normally functioning anti-idiotypic network exists and that there is an auto-anti(cid:173)
`idiotypic route to anti-receptor antibodies.
`Anti-receptor MAbs have been successfully generated by the two-step anti(cid:173)
`idiotypic approach which consists of the isolation of an appropriate anti-ligand
`antibody followed by immunization with the purified anti-ligand antibody. A critical
`success factor for the two-step approach is that the anti-ligand antibody (Abl) used
`for immm1ization should mimic the receptor as closely as possible. If an anti-idiotypic
`antibody that binds to the ligand binding site of the receptor is desired, an Ahl that
`binds to the receptor binding site of the ligand must be identified. This is particularly
`difficult for macromolecular ligands that possess multiple epitopes, onJy one of which
`reacts with the receptor binding site. In practice, the identification of an Abl as a
`surrogate receptor can be time-consuming and is often left to chance. The generation
`of the appropriate Ab2 also relies on coupling of the ligand at a site that preserves its
`specificity for the receptor binding site. Despite the successes reported with the two(cid:173)
`step approach (reviewed by Strosberg, 1989), accounts of failures have occasionally
`reached the literature, for example, the failure to generate anti-02 dopamine receptor
`antibodies (Abbott & Strange, 1986) and anti-aldostcrone receptor antibodies
`(Lombcs et al., 1989a).
`The feasibility of the one-step auto-anti-idiotypic approach to anti-receptor
`MAbs was first reported by Cleveland et al. (1983) who obtained anti-nicotinic
`acctylcholine receptor MAbs by immunizing mice with a structurally-constrained
`nicotinic receptor agonist. In addition to the lack of need to purify receptor, the one(cid:173)
`step approach offers a further time-saving advantage that the choice of the appropriate
`anti-ligand antibody is left to the idiotypic network rather than the experimenter.
`Other successes with this approach include the development of MAbs against the
`glucocorticoid (Cayanis et al., 1986), adenosine (Ku et al., 1987), aldosterone
`(Lombes et al., 1989b) and thyrotropin (Taub et al., 1992) receptors.
`The major objective of the present study was to screen MAbs raised by an
`auto-anti-idiotypic approach for CORP receptor binding properties. The development
`of anti-CORP receptor MAbs by the conventional approach of immunization with
`purified receptor was pursued by Wimalawansa ( 1992). The MAbs were initially
`
`133
`
`

`

`screened by ELISA with immobilized receptor purified from porcine cerebellum and
`had not been screened for receptor binding using rat or human tissues. Thus a further
`objecHve of the study was to screen these anti-porcine CGRP receptor MAbs for
`cross-reactivity with the rnt and human CGRP receptors.
`
`6.2. Methods
`
`6.2.1. Source of potential anti-receptor MAbs studied
`
`The MAbs studied in the present investigation come from two sources. Dr. C.
`Plumpton (Clinical Pharmacology Unit. Cambridge) provided 13 MAbs which were
`produced using an auto•anti-idiotypic approach. The MAbs were raised in mice which
`were immunized with RcxCGRP (described in Chapter 3) and cloned twice on the
`basis that the antibodies do not bind Rcx.CGRP but do bind affinity-purified rabbit
`P Abs against Rex.CG RP. These MAbs were coded Id 1 to Id 13 and were all isotyped as
`lgM. The ld MAbs used in the present study had been affouty purified by the use of
`an anti-mouse K light chain MAb column.
`Dr. S. Wimalawansa (Department of Medicine and Chemical Pathology, Royal
`Postgraduate Medical School, London) provided 5 MAbs which were raised against a
`CGRP receptor purified from porcine cerebellum (Wimalawansa, 1992; Wimalawansa
`et al., l 993). These MAbs were coded with the prefix RCG. Hybridoma supernatants
`were used in the present study.
`
`6.2.2. Receptor binding studies
`
`Materials
`
`2-[ !251]-iodohistidyl I 0-Ha.CGRP
`
`SK-N-MC cells
`
`Specific activity 2000 Ci/mmol (Amarsham )
`A TCC No. HTB I 0
`
`Binding buffer (rat brain and SK•N-MC cells) 1
`Bacitracin
`BSA
`MgCl2.6H20
`Tris I-ICI (pl-I 7.4)
`
`0.25%
`5mM
`50mM
`
`0.625 mg/m l
`
`1The binding buffer u~ed was similar lo that used for the liver membrane binding assay witJ1 the
`omission ofaprotinin and PMSF.
`
`134
`
`

`

`Wash buffer
`MgCl2.6H20
`Tris HCI (pH 7.4)
`
`5mM
`
`SOmM
`
`6.2.2.1. Experimental procedures
`
`6.2.2.J.J. Rat liver membrane preparatio11
`
`Rat liver membrane was prepared and binding assay performed according to the
`methods described in Chapter 5.
`
`6.2.2.J.2. Rat ,vl,o/e brain membra11e preparatio11
`
`Eight Sprague-Dawley rats weighing approximately 300 g were srunned and
`guillotined. The whole brain was dissected out and kept in saline on ice. These were
`weighed in a 50 ml homogenizing tube and homogenized in approximately 50 ml
`buffer (50 mM Tris HCI pH 7.4, 5 mM MgCl2). The homogenate was diluted to a
`total 20 volumes of buffer (i.e. 20 ml/kg), mixed on ice, and centrifuged at 48,000 g
`(20,000 rpm) at 4°C in a RC5C Sorvall Instruments (Dupont) centrifuge. The
`supernatant was discarded, the pellet resuspended in 20 volumes of buffer and the
`centrifugation repeated. The above step was repeated, giving 3 identical spins in all.
`The final resuspension was made in 2 volumes of buffer. One ml aliquots of the
`membrane suspension was stored at -20°C. Protein concentration was determined by
`the Biorad protein assay (see Chapter 4).
`
`6.2.2.J.3. SK-N-MC l111ma11 11euroblastoma cell membra11e preparatio11
`
`SK-N-MC cells were harvested, spun down and the pellet weighed (approximately
`0.25 g). Cells were then homogenized with 20 strokes of a glass-Teflon homogenizer
`at 650 rpm in 20 volumes (i.e. 20 ml/g) of 5 mM Tris HCI pH 7.4. The homogenate
`was cenLrifuged at 48,000 g (20,000 rpm) for 20 minutes at 4°C in a RC5C Sorvall
`centrifuge. The supernatant was discarded, the pellet resuspended as above in 20
`volumes of 5 mM Tris HCl pH 7.4 and left on ice for I hour to allow cell lysis. The
`above spin was then repeated. The pellet was resuspended in 20 volumes of buffer
`(50 mM Tris HCI pH 7.4, 5 mM MgCl2) and the membrane preparation stored in
`aliquots at -20°C.
`
`135
`
`

`

`6.2.2.1.4. Binding assay (rat wltole brain or SK-N-MC cell membra11e preparatio11)
`
`Whatman GF/C filters were soaked in 0.5% polyethyleneimine for at least 2 hours
`prior to filtering. The appropriate volume of rat bra.in membrane suspension was
`thawed, re-homogenized using a glass-Teflon homogenizer, and diluted to 2.5 mg/ml
`with 50 mM Tris HCI buffer pH 7.4 containing 5 mM MgCl2. SK-N-MC cell
`membranes were thawed, re-homogenized and used undiluted (approximately 100-
`200 µg protein per tube).
`The assay volume was 250 µI . Twenty-five µl of unlabelled CORP was added
`to each tube to give final concentrations of 10-12 to 10-7 M. Tubes for total binding
`contained assay buffer only. Non-specific binding tubes contained I o-7 M of
`unlabelled CGRP. Twenty-five
`µI
`of
`2-[ 1251]-iodohistidyl 10.Ha.CGRP
`(approximately 36,000 cpm) was added to each tube to give a final concentration of
`40 pM. The appropriate volume of assay buffer was added. One hundred µl of
`membrane suspension (250 µg protein for rat brain) was added to each tube. The tubes
`were vortex mixed and the assay mixture incubated for 60 minutes at room
`temperature.
`For the screening of antibodies, 50 µI Id MAb solutions were added to the
`polypropylene tubes (Starstedt) to give final concentrations of 15 to 60 µg/ml. r◄ ifty
`µl RCG MAb culture supernatants were tested. In some experiments MAbs were
`incubated with membranes for 5 days at 4 °C prior to addition of 2-[ 12511-
`iodohistidyl 10.Ha.CGRP and further incubation for 1 hour at room temperature. No
`difference in total and non-specific binding was observed after the 5 day incubation of
`the membrane suspension at 4°C compared with freshly
`thawed membrane
`suspension.
`Samples were .filtered through the pre-soaked GF/C filters on a Brandel 24-
`well cell harvester, followed by three washes with 3 ml ice-cold wash buffer. Filter
`paper was placed into counting tubes and counted for 1 minute in a LKB Wallac 1272
`Clinigamma counter with four 1.5 inch detectors.
`
`6.2.3. lmmunocytochemistry
`
`6.2.3.1. Principles
`
`The immunocytochemical method used in the present study was based on the Avidin(cid:173)
`Biotin Complex (so-called ABC) system. The immunoperoxidasc staining technique
`employed an unlabelled primary antibody, followed by a biotinylated secondary
`antibody and then a pre-formed avidin and biotinylated horseradish peroxidase
`macromolecular complex.
`
`136
`
`

`

`Avidin is a large glycoprotein from egg white which has a very high affinity
`(four binding sites per molecule) for biotin, a vitamin of low molecular weight found
`in egg yolk. Biotin can be coupled to antibody in high molecular proportion, or to a
`label such as peroxidase. Avidin may also be labelled with, for example, peroxidase
`or fluoresccin. The complex of avidin and biotinylated peroxidase are reacted together
`in such proportion that some biotin-binding sites on the avidin molecule are not fi1 led
`by biotinylated peroxidase, but are free to react with the biotin of the second antibody.
`The substitution for avidin with streptavidin, derived from Streptococcus
`avidini, offers some advantages. The streptavidin molecule is uncharged relative to
`animal tissue, unlike avidin which has an isoelectric point of 10, and therefore
`electrostatic binding to tissue is eliminated. In addition, streptavidin does not contain
`carbohydrate groups which might bind to tissue lectins. A streptavidin-biotinylated
`horseradish peroxidase complex was evaluated in the present study.
`The Vectastain® Elite ABC kit eventually chosen in the present study
`contained Avidin DH and biotinylated horseradish peroxidase H reagents which have
`been prepared to form ideal complexes for immunoperoxidase staining.
`
`Materials
`
`Nonnal horse serum
`
`Anti-mouse K light chain MAb I 87. I (biotinylated by C. Plumpton)
`
`Biotiny)ated horse anti-mouse lgG (H + L; rat adsorbed; BA-200 I)
`Purified mouse lgM, K from TEPC tumour line (M-2770)
`Affinity-purified rabbit anti-CG RP PAbs
`Strept.avidin-biotinylated horseradish peroxidase complex (RPN I 051)
`Fluorescein-streptavidin (RPN 1232)
`Vcctastain® £lite Aac kit
`3,3' Oiaminobenzidine (DAB)
`Hydrogen peroxide solution 30%
`Paraformaldehyde
`Xylene
`DEPEX mounting medium2
`Propidium iodide
`CitiFluor aqueous mountant
`Triton-X 100 (10% stock in PBS)
`
`Supplier
`
`Vector Laboratories,
`Peterborough
`
`European Collection of
`Animal Cell Cultures,
`Porton Down
`
`Vector
`
`Sigma
`
`C. Plumpton
`Amcrsham
`
`Amersham
`Vector
`
`Amersham
`BDH
`BDTI
`
`B011
`
`BDll
`
`Molecular Probes
`City University, London
`Sigma
`
`2DEPEX is named for its components, 10 g distrcne 80, 5 ml dibutyl phthalate, and 35 ml ofxylene
`
`137
`
`

`

`Reagen ls
`0.2 M Phosphate buffer
`Solution A:
`31.2 g sodium di hydrogen orthophosphate in I 000 m Is of deionized H20.
`Solution B:
`28.3 g disodium hydrogen orthophosphate in IOOO mis of deionized T120.
`9.5 ml of A+ 405 ml of B, made up to 500 ml with deionized H20,
`
`4% Paraformaldehyde
`Paraforma1dehyde
`
`80 g
`
`Deionized H20
`500ml
`(Heated witl1 stirring to 60°C, held for 5 minutes, and I M NaOH added dropwisc to clear solution.)
`The above solution was cooled and added to 500 ml 0.2 M phosphate buffer. The pH of the solution
`was adjusted to 7.4 with HCI.
`
`Peroxidase st1bstrate so/111/on (prepared just prior to use)
`DAB
`25 mg
`Hydrogen peroxide (30%; I 00 volumes)
`0.1 M PBS/0.3% Triton-X I 00 (PBSTx)
`
`16 µI Gust prior lo use)
`JOO ml
`
`Vectastain® Elite ABC reagent
`Reagem A
`2 drops (from bottle supplied)
`Reagent B
`2 drops
`0.1 M PBS pH 7.4
`5ml
`The solution was mixed immediately and allowed to stand for 30 minutes prior to use.
`
`Gelatin-subbing of slides
`Glass slides were immersed in Decon detergent solution overnight, rinsed and dried in an oven. Six
`grams of gelatin was dissolved in 1200 ml deionized water by stirring and heat. When dissolved, 0.6 g
`of chromic potassium sulphate was added and slides immersed in this solution for 5 minutes. Slides
`were then dried in an oven.
`
`6.2.3.2. Transcardiac perfusion fixation
`
`Fixation protocols must (1) prevent antigen leakage, (2) permeabilize the cell to allow
`access of the antibody, (3) keep the antigen in such a form that it can be recognized
`efficiently by the antibody, and (4) maintain the cell structure. Transcardiac perfusion
`is the method of choice for the preservation of central nervous system tissue. It aids
`fixation by allowing excellent and quick penetration of fixative and thus prevents a
`number of artefacts associated with the far slower immersion fixation of tissues.
`Paraformaldehyde is a cross-linking reagent which form intermolecular bridges,
`
`138
`
`

`

`,
`
`normally through free amino groups, thus creating a network of linked antigens.
`However, such fixation may denature protein antigens.
`
`6.2.3.2.1. Experimental procedllres
`
`Each Sprague-Dawley rat was deeply anaesthetized by i.p. injection of pentobarbitone
`sodium (Rhone-Merieux). The chest cavity was opened to expose the heart. The
`descending aorta was clamped and the perfusion needle inserted at the apex of the
`heart into the left ventricle. Once the needle was inserted, the right atrium was cut to
`allow irrigation of the upper vasculature of the animal. The rat was perfused with
`0.9% saline containing 2S units/ml heparin al 100 mmHg pressure (approximately
`circulation pressure) until the fluid leaving the atrium appeared clear (approximately
`300 ml). This was followed by perfusion with freshly prepared 4% paraformaldchyde
`in 0.1 M PBS pH 7.4 until the upper body of the animal appeared pale and was rigid
`(approximately 700 ml). The entire spinal column was then dissected and immersed in
`4% parafonnaldehyde at 4°C overnight. The spinal cord was removed the next day
`and immersed overnight in 30% sucrose in 0.1 M PBS pH 7.4 at 4°C.
`
`6.2.3.3. Snap freezing of tissues
`
`rt is important that tissue is frozen rapidly to prevent the formatjon of ice crystals
`(from water within the tissue) which will damage the tissue. Tissues were snap-frozen
`in isopentaoe at -35 to -40°C. This was achieved by cooling a beaker of isopentaoc on
`dry ice and monitoring the temperature of isopentane with a temperature probe. At
`this temperature, the white and grey matter of tissues from the central nervous system
`freeze at the same rate, thus minimizing any cracking of' tissue. Frozen tissue were
`used fresh or stored at -70°C
`
`6.2.3.4. Cryostat sections
`
`In experiments involving the use of free-floating sections, 30 µm sections were cut
`from the rat spinal cord (L4-L5) at -20°C using a 2800 Frigocut E cryostat (Reichert(cid:173)
`Jung) or a Bright OTF/AS cryostat (Bright Instrument, Huntingdon). Sections were
`immediately immersed in 0.1 M PBS/0.3% Triton-X 100 (PBSTx) in permeable
`plastic capsules (Agar Scientific Ltd., Stansted) placed within the wells of a 24-well
`tissue culture plate (CoStar). Two spinal cord sections were placed into each well.
`
`139
`
`

`

`6.2.3.5. Immunocytochemical staining of free-floating tissue sections
`6.2.3.5.1. Optimization of staining procedure
`
`The immunostaining procedures described below were optimized by systematically
`investigating the effect of several experimental variables on the staining of adjacent
`tissue sections. The optima] concentration of primary antibody was determined by
`dilution studies. Two secondary antibodies were compared: biotinylated anti-mouse K
`light chain MAb 187.1 and the Vector biotinylated horse rat-absorbed anti-mouse (gG
`which exhibits cross-reactivity with mouse IgM. Streptavidin-biotinylatcd horseradish
`peroxidase complex from Amersharn (diluted I in 200 with PBSTx) and the
`Vcctastain® Elite ABC reagent were compared. The concentration of DAB was
`compared at 0.025% and 0.05%.
`
`6.2.3.5.2. Experime11tal procedures
`
`All procedures were carried out at room temperature with the exception of overnight
`incubation with primary antibody at 4°C. fncubation and washing steps were
`performed with gentle shaking of the tissue culture plate on an orbital tray shaker. Tbe
`washing step consisted of reagent removal followed by 3 incubations with 300 µl
`fresh PBSTx over 5 minute intervals.
`PBSTx was removed from wells containing free-floating sections by the use of
`a Pasteur pipette attached to a vacuum. Four hundred µl of 0. t M PBS containing
`normal horse serum 3%, 0.1% BSA and 0.3% Triton-X 100 (blocking reagent) was
`added to each well. Tissue sections were incubated with blocking reagent for 1 hour.
`After removal of the blocking reagent, tissue sections were incubated overnight at 4°C
`with 300 µl of primary antibody appropriately diluted in blocking reagent (15 to
`60 µg/ml for Id MAbs). RCG MAb supcmatants were tested undiluted. Tissue
`sections were washed and incubated for 90 minutes with 300 µI of biotinylated
`secondary anti-mouse antibody at a dilution of 1 in 200 (in PBSTx). This was
`followed by washing and incubation for 60 minutes with 300µ1 of Vectastain® Elite
`ABC reagent. Alter washing, 300 µl substrate solution (DAB solution 0.025%
`containing 0.005% hydrogen peroxide in PBSTx) was added and the development of
`colour monitored under a microscope (Wild Hcerbrugg; Leitz Instruments Ltd.). The
`reaction was stopped by washing twice in deionized water.
`Tissue sections were mounted on glass slides (non-gelatinized; Chance
`Propper Ltd., Smethwick, Warley) with the aid of a brush and allowed to air dry.
`Slides were sequentially immersed in 70% ethanol (5 minutes), absolute ethanol (5
`minutes), and finally in xylene (5 minutes). Coverslips (Chance Propper Ltd) were
`mounted with the non-aqueous mounting medium DEPEX.
`
`140
`
`

`

`6.2.3.5.3. Experimental co11tro/s
`
`Method specificity was detennined by the use of omission controls. Primary antibody,
`biotinylatcd secondary antibody, streptavidin-biotinylated horseradish peroxidase
`complex and DAB were omitted in control tissue sections in order to identify the
`source of potential non-specificity.
`Antibody specificity was investigated by the use of a non-specific mouse lgM
`as a control antibody and testing whether pre-absorption with antigen could diminish
`immunostaining. Having dctennincd the lowest concentration of primary antibody
`compatible with good immunostaining, Id MAbs were pre-absorbed with affinity(cid:173)
`purified rabbit anti-CORP PAbs (10 to 50 times excess molar concentration)
`overnight prior to use in immunostainiog of tissue sections.
`The most relevant question in U1e present study was whether immunostaining
`with Id MAbs could be attributable to CORP receptor localization. CORP itself
`should inhibit immunostaining if CORP receptors were localized by ld MAbs.
`Therefore, tissue sections were pre-incubated with excess Ra.CORP (10 µM) in
`binding assay buffer or binding assay buffer alone, blocked with normal horse serum
`(blocking reagent above), followed by overnight co-incubation of Id MAbs with
`10 ~tM Ra.CGRP.
`
`6.2.3.6. Immunocytochemistry using fresh (unfixed) tissue sections
`
`The immunostaining of fresh, unfixed, spinal sections was tested because of the
`possibility
`that paraformaldehyde fixation denatures
`the CORP receptor. If
`immunostaining of fresh tissue sections was possible, it would allow testing of the
`specificity ofld MAbs for the CORP receptor by displacement with excess CGRP.
`The immunocytocbemicaJ procedure used was similar to that described above
`for fixed tissue sections. However, tissue sections were mounted on slides and thinner
`14 µm sections were used to facilitate antibody penetration. In addition, Triton-X I 00
`was avoided in all steps.
`
`6.2.3.7. Immunocytochemistry of cultured cells
`
`Immunostaining of live and fixed cells was attempted in order to answer the major
`question of whether ld and RCO MAbs specifically localize the CORP receptor.
`Specificity could be assessed by studying cells previously shown by receptor binding
`stuclies to be positive or negative for CORP receptors. Immunostaining live cells
`would avoid the potential denaturing effect of paraformaldehyde fixation and allow
`the performance of displacement stuilies with CORP. In addition, higher resolution
`
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`may be obtained and 3-dimensional views obtained by confocal fluorescence
`microscopy could identify whether a membrane protein is immunostained (e.g., see
`Ornatowska & Glascl, 1992).
`The SK-N-MC human cell line clearly expresses CORP receptors, as shown in
`the present study and by others (Semark et al., 1992). However, specific CGRP
`binding could not be demonstrated in a related cell line, SK-N-SH. This cell line was
`therefore used as a negative control. A major interest was to use anti-receptor MAbs
`as pharmacological tools in the rat. Therefore, the rat. L6 myocyte cell line which
`expresses high affinity CGRP receptors was investigated. Specific binding of CGRP
`to intact 16 myoeytes has been demonstrated (Poyner el al., 1992). The AR42J rat
`exocrine pancreas cell line which does not express CGRP receptors (Poyner el al.,
`1992) was used as a negative control.
`
`6.2.3. 7.1. Cell culture
`
`SK-N-MC (ATCC No. HTB 10) and SK-N-SH (ATCC No. HTB 11) human
`neuroblastoma cells were obtained from the American Type Culture Collection. L6 rat
`thigh muscle cells (ECACC No. 85011421) and AR42J rat exocrine pancreas ceJls
`(ATCC No. CRL 1492) were obtained from the European Collection of Animal Cell
`Cultures and Flow Laboratories respectively. Cells were cultured in media specified
`by the suppliers and grown to about 80% confluence for immunostaining.
`
`6.2.3. 7.2. Coati11g of coverslips wit/, Poly-L./ysi11e
`
`Cells were grown on poly-L-lysine coated covcrslips within wells of 12-well tissue
`culture plates for the purpose of immunostaining. Poly-L-lysine binds to most solid
`supports through its charged side chains. The positively charged polymer provides a
`site for binding of cells (which carry a overall negative charge). Although this cross(cid:173)
`link is not covalent, it is sufficiently strong for most cell staining techniques.
`The following procedures were performed in a laminar flow cabinet. Poly-L(cid:173)
`lysine (Sigma) was dissolved in sterile water to 5 µg/ml. Coverslips (18 mm diameter)
`were placed into wells of a 12-well tissue culture plate. One ml poly-L-lysine was
`added to each well. After an incubation period of 30 minutes, the solution was
`aspirated and coverslips washed with I ml sterile water. The eoverslips were allowed
`to dry before the seeding of cells.
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`6.2.3. 7.3 Experimental procedures
`
`The immunostaining procedures were performed in 12-well tissue culture plates at
`room temperature. Except for the final wash, each washing step consisted of reagent
`removaJ followed by 3 incubations with fresh 0.1 M PBS over 5 minute intervals.
`Cells were removed from the incubator, washed in DMEM medium (with
`25 mM HEPES and 4.5 g glucose but without pyruvate [Gibco]) for 5 minutes,
`followed by incubation in the same medium containing 3% normal horse serum
`(blocking reagent) for 60 minutes. The blocking reagent was removed and cells
`incubated with Id MAbs diluted in blocking reagent (15 to 60 µg/ml) for 60 minutes.
`RCG MAb supernatants were tested undiluted. A control without primary antibody
`was included. After washing, cells were incubated with biotinylated anti-mouse
`secondary antibody (Vector) diluted 1 in 200 in 0.1 M PBS for 60 minutes. The cells
`were washed and incubated for 60 minutes with fluorescein-streptavidin diluted I in
`200 with 0.1 M PBS. After a final wash in PBSTx, cells were incubated with the
`nuclear counterstain propidium iodide (t mg/ml stock; diluted l in 5000 with PBSTx)
`for 30 seconds. A small drop of aqueous mountant (Citifluor) was added to glass
`slides for the mounting of coverslips. A coverslip with adhered cells was gently
`apposed to the aqueous mountant on a glass slide and allowed to dry for a short
`period. Finally, the coverslip was sealed and fixed in position with clear nail varnish.
`lmmunostaining of fixed cells was also performed. Fixation was performed by
`washing cells with 0.1 M PBS followed by incubation with 4% paraformaldehyde in
`0.1 M PBS for 10 minutes. After washing, the same procedures described above were
`followed.
`Jmmunoperoxidase staining using the same procedures described for tissue
`sections was also attempted.
`
`6.2.3.8. Microscopy and Photography
`
`Tissue sections were viewed under a transmitted light microscope (Leitz Dialux
`20EB, Germany). Photography was perfonned a Wild Photoautomat MPS 51S/45
`camera at-tached to the microscope. A rare earth dydidium filter was placed over the
`1 ight source and the light condenser was used except in lowest power. EPT-160T
`(Kodak Ektachrome; 160ASA, tungsten) slide film was used.
`For immunofluorescence experiments, a drop of immersion oil was placed on
`top of the coverslip and the slide viewed under a fluorescence microscope (Leitz;
`Filter I; the wavelength for maximal excitation of fluoresccin is 495 run).
`
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`6.2.3.9, Image Analysis
`
`Image analysis of Id MAb immunostained sections was performed to assess whether
`the intensity of cell staining corresponded to the expected distribution of receptors as
`assessed by receptor autoradiography.
`The MCID image analysis system (Microcomputer Imagi11g Device; Imaging
`Research Inc., Brock University, Ontario, Canada) was used. The system consisted of
`a PC-compatible computer running under the OS/2 operating system, an imaging
`system and software. A Compaq